It is often necessary to detect and/or quantify negatively charged analytes in various samples, such as biological samples. Of the negatively charged analytes, oligonucleotides are of a particular interest to the pharmaceutical industry as therapeutic agents. For example, oligonucleotides may be used therapeutically as sense/antisense deoxyribonucleic acid (DNA) oligonucleotides or as interfering ribonucleic acid (RNAi) oligonucleotides to inhibit proteins, or as nucleic acid based aptamers (McGinnis et al., Journal of Chromatography B 2012; 883-884:76-94; Agrawal and Zhao, Current Opinion in Chemical Biology 1998; 2:519-28; Lee et al., Current Opinion in Chemical Biology 2006; 10:282-9). This renewed interest has, in part, been fueled by the commercial success of therapeutic oligonucleotides, such as Formivirsen (Perry and Balfour, Drugs 1999; 57:375-80) and Pegaptanib (Gragoudas et al., New England Journal of Medicine 2004; 351:2805-16), in the treatment of cytomegalovirus retinitis and age-related macular degeneration, respectively.
Conceptually, synthetic therapeutic oligonucleotides are comprised of their corresponding base nucleic acids and are synthesized with fragment lengths typically ranging from 15 nucleotides (15′ mer or 15 nt) and 30 nucleotides (30′ mer or 30 nt), although fragments with more than 30 nt have also garnered increasing interest from the pharmaceutical industry (McGinnis et al., Journal of Chromatography B 2012; 883-884:76-94). Chemical modifications to the phosphodiester backbone are commonly incorporated into synthetic oligonucleotides to increase their stability in vivo against endo- and exonucleases, as well as improve efficacy through increased cellular uptake and binding (McGinnis et al., Journal of Chromatography B 2012; 883-884:76-94; Akhtar et al., Life Sciences 1991; 49:1793-801; Vaerman et al., Blood 1997; 90:331-9). While the process for oligonucleotide synthesis is well-controlled, several factors can alter the end product. Purity of the starting materials can affect sequence failure rate, while random insertions and deletions can result in improper sequence generation (Gilar and Bouvier, Journal of Chromatography A 2000; 890:167-77; Gilar, Analytical Biochemistry 2001; 298:196-206). Furthermore, base pair switches and the presence of chiral centers can result in difficulties in separating isomers and diastereomers, respectively (Dias and Stein, Molecular Cancer Therapeutics 2002; 1:347-55). With over a hundred therapeutic oligonucleotides currently in development or in clinical trials, factors such as safety, efficacy, and stability are leading concerns for pharmaceutical companies and regulatory agencies (McGinnis et al., Journal of Chromatography B 2012; 883-884:76-94). In this regard, analytical methods for robust and accurate detection and quantification of negatively charged analytes, such as oligonucleotides, are highly desirable.
The intrinsic negative charge of the phosphodiester backbone present in oligonucleotides, combined with their ultraviolet (UV) absorbance properties has made chromatographic based characterization methods, such as ion-exchange chromatography (IEC) and ion-pairing reversed phase chromatography (IP-RPLC), popular choices in the characterization of oligonucleotides (Waters et al., Journal of Clinical Oncology 2000; 18:1812-23; Arora et al., Journal of Pharmaceutical Sciences 2002; 91:1009-18; Bunček et al., Analytical Biochemistry 2006; 348:300-6; Huber et al., Analytical Biochemistry 1993; 212:351-8; Apffel et al., Analytical Chemistry 1997; 69:1320-5; McCarthy et al., Analytical Biochemistry 2009; 390:181-8).
Charge-based separations, such as anion exchange chromatography, are well suited for characterization of oligonucleotides containing N-X deletions, however, oligonucleotides containing apurinic sites, base inversion isomers, and other base modifications are not readily characterized using ion exchange chromatography (IEC; McGinnis et al., Journal of Chromatography B 2012; 883-884:76-94). Furthermore, buffers and salt gradients typically used in IEC prevent the coupling of IEC to mass spectrometry (MS), which may be useful as a complementary orthogonal technique for characterizing oligonucleotides containing difficult to analyze base modifications. Analytical techniques, such as ion-pairing reversed phase liquid chromatography (IP-RPLC), have become popular for characterizing oligonucleotides, in part due to their compatibility with MS based techniques. This was demonstrated by Apffel and colleagues using triethylamine (TEA) as the IP base buffered in hexafluroisopropanol (Apffel et al., Analytical Chemistry 1997; 69:1320-5; Apffel et al., Journal of Chromatography A 1997; 777:3-21). Oligonucleotides may be separated with high separation efficiency using hydrophobic bonded phases with adsorbed n-alkyl IP reagents, such as amines, based on charge interactions of the phosphodiester backbone and, to a lesser degree, the secondary structure of the oligonucleotide and hydrophobicity of the base nucleotides (Gilar, Analytical Biochemistry 2001; 298:196-206; Huber et al., Analytical Biochemistry 1993; 212:351-8; Gilar et al., Journal of Chromatography A 2002; 958:167-82; Dickman, Journal of Chromatography A 2005; 1076:83-9). MS based methods can provide accurate mass information for oligonucleotides and are highly desirable for analyses that require high sensitivity, such as toxicology and metabolite studies, including determination of pharmacodynamics and pharmacokinetic parameters (Dias and Stein, Molecular Cancer Therapeutics 2002; 1:347-55; Huber et al., Analytical Biochemistry 1993; 212:351-8; Zhang et al., Analytical Chemistry 2007; 79:3416-24; Deng et al., Journal of Pharmaceutical and Biomedical Analysis 2010; 52:571-9; Beverly et al., 2005; 19:1675-82). There are, however, challenges associated with the MS based techniques for the analysis of negatively charged analytes, such as oligonucleotides (Lin et al., Journal of Pharmaceutical and Biomedical Analysis 2007; 44:330-41; Cech and Enke, Mass Spectrometry Reviews 2001; 20:362-87; Keller et al., Analytica Chimica Acta 2008; 627:71-81; Ende and Spiteller, Mass Spectrometry Reviews 1982; 1:29-62).
One of such challenges involves contamination of the analytical system with alkali metal ions. There are several possible ways for the analytical system to become contaminated with alkali metal ions. For example, alkali metal oxides are used in the manufacturing process of laboratory glassware, such as borosilicate glassware, and can leach into solvents over time in the presence of acids, bases and organic solvents (Varshneya, Fundamentals of inorganic glasses, Elsevier, 2013). Similarly, metal surfaces throughout the fluidic path can potentially leach metal ions via corrosion that occurs when the metal surfaces are exposed to acids and bases commonly used in LC separations. Alternatively, the impurities present in the solvents and reagents can also contribute to adduct formation in LC/ESI-MS based separations.
Electrospray ionization (ESI) MS based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation (Apffel et al., Analytical Chemistry 1997; 69:1320-5; Zhang et al., Analytical Chemistry 2007; 79:3416-24; Huber et al., Analytical Chemistry 1999; 71:3730-9). Positively charged cations of alkali metal salts, such as sodium (Na+) and potassium (K+), are electrostatically attracted to the negatively charged polyanionic backbone of oligonucleotides (Muddiman et al., J. Am. Soc. Mass Spectrom. 1996; 7:697-706; Cheng et al., Analytical Chemistry 1995; 67:586-93). Alkali metal adducts, which can occur singly, multiply, or as any combination as shown in Table 1 below, directly impact the sensitivity of MS based analyses, because the available charge is distributed across the parent ion and the adducts. This problem becomes further compounded for longer oligonucleotides, because length of the sequence, the number of observed charge states, and base modifications can impact the degree of adduct formation and spectral complexity (Fountain et al., Rapid Communications in Mass Spectrometry 2003; 17:646-53; Gong and McCullagh, Rapid Communications in Mass Spectrometry 2014; 28:339-50).
TABLE 1Formula for predicting charge states of alkali metal adducts of oligonucleotides analyzed in negative scan mode using ESI-MS. Molar masses of hydrogen (H), sodium (Na), and potassium (K) are 1.008 g/mol, 22.9898 g/mol and 39.0983 g/mol, respectively.Adduct Ion CompositionPredicted m/z Value[M − H]−M − 1[(M − H) + Na]−(M − 1) + 23[(M − H) + K]−(M − 1) + 39[M − xH]−X(M − xH)/x[(M − xH) + yNa]−X[(M − xH) + 23y]/x[(M − xH) + yK]−X[(M − xH) + 39y]/x[(M − xH) + yNa + zK]−X[(M − xH) + 23y + 39z]/x
Current strategies for reducing the extent of alkali metal adducts formation during oligonucleotide analysis with ESI-MS included the use of offline and online desalting procedures with varying success. Offline desalting procedures that incorporate the use of hydrophobic resins, molecular weight cutoff filters, and solid phase extraction techniques have been shown to be effective in reducing adduct formation (Gilar and Bouvier, Journal of Chromatography A 2000; 890:167-77; Ragas et al., Analyst 2000; 125:575-81; Bayer et al., Analytical Chemistry 1994; 66:3858-63; Deroussent et al., Rapid Communications in Mass Spectrometry 1995; 0:1-4; Jiang and Hofstadler, Analytical Biochemistry 2003; 316:50-7). However, the additional sample preparation steps required are not readily amendable to high-throughput platforms. Online desalting strategies have included incorporation of microdialysis or cation exchange chromatography (Muddiman et al., Analytical Chemistry 1996; 68:3705-12; Huber and Buchmeiser, Analytical Chemistry 1998; 70:5288-95). These techniques, while more amendable to high throughput methods, can increase instrument configuration complexity and require additional method re-conditioning/equilibration steps which can impact productivity. A more appealing alternative to reducing alkali metal adducts in oligonucleotide analyses has been to use sample additives that act as cation scavengers or work to suppress adduct formation via displacement mechanisms. For example, Limbach and colleagues observed that the addition of trans-1,2-cyclohexanediaminetetraacetic acid monohydrate (CDTA), a metal chelator, reduced adduct formation in the analysis of RNA (Limbach et al., J. Am. Soc. Mass Spectrom. 1995; 6:27-39). Alternatively, addition of base, such as piperidine or TEA, was found to suppress adduct formation (Muddiman et al., J. Am. Soc. Mass Spectrom. 1996; 7:697-706; Cheng et al., Analytical Chemistry 1995; 67:586-93; Greig and Griffey, Rapid Communications in Mass Spectrometry 1995; 9:97-102). In contrast to Limbach et al., an extensive study by Gong and McCullagh of IP reagents buffered with HFIP found that metal chelators, such as CDTA and ethylenediaminetetraacetic acid (EDTA), did not have a significant impact on adduct formation (Gong and McCullagh, Rapid Communications in Mass Spectrometry 2014; 28:339-50). Their work indicated that adduct formation was dependent on oligonucleotide size. Interestingly, with the exception of the 10 nt polyT sequence, more than 25% of the MS signal was in a metal adduct form. Despite these conflicting reports, the relevance of suppressing cation adduction is evident in the diversity of strategies employed across instrument configurations and experimental settings.
These approaches, while effective in reducing metal adduct formation, do not address contribution of the instrument to metal salt adducts formation, a challenging task considering the ubiquitous nature of alkali metal salts in LC separations (Keller et al., Analytica Chimica Acta 2008; 627:71-81; Ende and Spiteller, Mass Spectrometry Reviews 1982; 1:29-62). Potential sources of metal adduct ions can be found throughout a conventional LC system configuration, as shown in FIG. 1. Glass surfaces such as the ones found in reservoir bottles and sample vials can contain trace amounts of alkali metal salts as a byproduct of the manufacturing process used to produce them. Leaching of these trace metal salts can occur in the presence of solvents, acids and bases (Varshneya, Fundamentals of inorganic glasses, Elsevier, 2013). Purity of the solvents used in chromatographic separations can also increase the concentration of salt ions present in an analytical separation. Similarly, the abundance of alkali metal salts in biological samples can also contribute to adduct formation in ESI-MS based analyses. Furthermore, the chromatography system itself can also act as a source of metal adduct ions as alkali metal salts are deposited on high surface area points of contact found throughout the system, such as mixers, filtering frits, and column frits. Therefore, an easy and robust method for reducing the amount of alkali metal salts present in an analytical system and for increasing the sensitivity of detection and/or quantification of a negatively charged analyte, such as an oligonucleotide, is needed.